The principle of hybridization is the property upon which most practical methods of detecting nucleic acid sequences are based. In general, methods for detecting the presence of particular target nucleic acid sequences involve employing a complementary sequence termed a probe, generally detectably labeled, and incubating this labeled probe sequence with the test sample thought to contain the target nucleic acid sequence. These complementary nucleic acid sequences hybridize to one another under suitable conditions to form probe-target hybridization complexes which can be identified through the presence of the detectable label. Various methods in which particular aspects of this basic process have been optimized for the purpose of addressing specific assay requirements have developed over time. (see Wetmur, 1991, Critical Reviews in Biochemistry and Molecular Biology 26, 227-259).
While some homogeneous phase hybridization assays permit detection of the hybridized probe-target complex without removal of excess unhybridized probe sequences present in the assay solution (see Tyagi and Kramer, 1996, Nature Biotechnology 14,303-308; Arnold, Waldrop, and Hanmmond, 1990, U.S. Pat. No. 4,950,613), very sensitive assays require separation of the hybridized complex from the unhybridized probe prior to the detection step. Many different methods have been employed to accomplish this separation, including several that rely on differences in physical characteristics between the two products. Other methods employ the use of a second nucleic acid sequence, described as a "capture" probe, for the purpose of separating the probe-target complex from the unhybridized probe. Capture probes are generally immobilized on a solid support and are selected to hybridize to a different portion of the target nucleic acid sequence than does the labeled probe. Thus, a tripartite capture probe-target-labeled probe complex forms which is bound to the solid support, while the unhybridized labeled probe remains unbound in solution, allowing the two products to be readily separated. Such assays are referred to as "sandwich" hybridizations (see, for example, Engelhardt and Rabbani, 1994, U.S. Pat. No. 5,288,609). Although widely used, these assays require a number of steps to perform and are quite time-consuming.
Alternative assay methods, termed displacement assays, were developed in an attempt to simplify the method of identifying nucleic acids. A schematic illustration of a standard displacement assay is depicted in FIG. 1 (see Diamond, S. E., et al., 1988, U.S. Pat. No. 4,766,062; Williams, J. I., et al. 1988, U.S. Pat. No. 4,766,064; Vary, 1987, Nucleic Acids Res. 15, 6883-6897; Vary et al., 1986, Clinical Chemistry 32, 1696-1701). In a standard displacement assay, a tether nucleic acid sequence, complementary to the target nucleic acid sequence, is hybridized to a shorter, detectably-labeled signal nucleic acid sequence, complementary to a specific subsequence of the tether sequence. The signal nucleic acid is fully base-paired with the tether nucleic acid in this signal-tether complex, but the longer tether nucleic acid retains a single-stranded region. Upon the introduction of a test sample containing the target nucleic acid, the target hybridizes to the single-stranded portion of the tether component. Since the target is homologous to the entire length of the tether, a homologous strand exchange reaction with the signal nucleic acid is initiated, and the target displaces the signal from the tether. This strand exchange reaction proceeds rapidly in the direction of signal nucleic acid displacement because the target is longer and forms a more stable duplex with the tether (see Green, C. and Tibbetts, C., 1981, Nucleic Acids Res. 9, 1905-1918). The amount of displaced labeled signal nucleic acid is measured to determine the amount of target nucleic acid in the sample. The tether component of the probe complex can also be linked to a solid support, so that separation of the solid and solution phases results in isolation of the signal nucleic acid.
Unfortunately, despite their advantages, standard displacement assays do have certain drawbacks. For example, when target nucleic acid hybridizes to a tether not hybridized to a signal nucleic acid, or when a displaced signal nucleic acid hybridizes to a tether nucleic acid which was not previously hybridized to a signal nucleic acid, a decrease in the detection signal produced per unit of target hybridized results. Moreover, if the hybridized complex is not stable, an undesirable background signal can be introduced, which complicates interpretation of the assay results and reduces the sensitivity of the assay.